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Abstract
Aim and Objective: The aim and objective of this study were to develop a spectrophotometric method for the
assay of selexipag (selective IP prostacyclin receptor agonist indicated for the treatment of pulmonary arterial
hypertension) in pure and pharmaceutical formulations so that it will be an alternative quantitative method to
chromatographic methods which require large quantities of organic solvents, where some are with hazardous and
toxic properties. Materials and Methods: The method is based on the diazo coupling of selexipag with diazotized
p-nitroaniline in alkaline medium to form a stable green-colored and water-soluble azo dye with a maximum
absorption at 510 nm. Optimization of reaction conditions was carried out to get highly sensitive and stable
colored complex. Results and Discussion: Beer’s law is obeyed over the concentration range of 2–12 µg/mL with
a molar absorptivity of 3.33 × 104 L/mol/cm. The limit of detection was 0.35 μg/mL and limit of quantification
was 1.0 µg/mL. The results demonstrated that the procedure is accurate, precise, and reproducible (relative
standard deviation <2%). Conclusions: This method was tested and validated for various parameters according
to the current ICH guidelines.
S
elexipag is a selective IP prostacyclin Analytical reagent grade chemicals were used throughout the
receptor agonist and suggested for investigation. Double distilled water was used, and solutions
the treatment of pulmonary arterial were freshly prepared. Absorbance was measured using double
hypertension to delay disease progression and beam UV-Visible Spectrophotometer (TECHCOMP, UV
reduce the risk of hospitalization.[1] Selexipag 2310) equipped with HITACHI software version 2.0. Quartz
is rapidly absorbed after oral administration cuvettes (10 mm path length). Samples were weighed using
and hydrolyzed to the pharmacologically Shimadzu AUX-220 balance. Spectroscopic measurements
more active metabolite ACT-333679.[2] It was were conducted at room temperature (25 ± 5°C).
synthesized by Nippon Shinyaku and later jointly
developed with Actelion Pharmaceuticals Ltd.
Preparation of Reagents
2-{4-[(5,6-diphenylpyrazin-2-yl)(propan-2-yl)
amino]butoxy}-N-methanesulfonylacetamide
Para nitroaniline (PNA) solution (7.24 × 10–3 M): Accurately
is the chemical name of selexipag [Figure 1].[3]
100 mg of PNA was weighed and was taken in a 100 mL
Thorough literature survey makes it clear
Address for correspondence:
that no visible spectrophotometric method
Dr. Venkata Nadh Ratnakaram, Department of Chemistry,
is reported so far for the determination of
GITAM University, Bengaluru Campus, Nagadenahalli,
selexipag, but only one high-performance
Doddaballapur Taluk, Bengaluru – 561 203, Karnataka,
liquid chromatographic (HPLC) method was
India. Phone: +91-9902632733.
published.[4] Therefore, the current study
E-mail: doctornadh@yahoo.co.in
reports the development and validation of a
flexible visible spectrophotometric method for
Received: 06-07-2018
the determination of selexipag in bulk drug and
Revised: 07-12-2018
tablet dosage formulations using a diazotized
Accepted: 16-12-2018
coupling reaction.
volumetric flask. It was dissolved in 20 mL of one normal solutions (PNA, acid, sodium nitrite, and sodium hydroxide),
HCl solution and made up to the mark with distilled water. time for the formation and stability of colored product, and
temperature. Variation of reaction conditions was carried out
Sodium nitrite solution (1.45 × 10–2 M): Accurately 100 mg of to optimize them. Color species absorbance was measured
NaNO2 was weighed and was taken in a 100 mL volumetric in the establishment of optimum conditions by changing the
flask. It was dissolved in distilled water and made up to the mark. condition of one parameter at a time and by maintaining fixed
conditions for others.
Sodium hydroxide solution (1 M): Accurately 4 g of NaOH
was weighed and was taken in a 100 mL volumetric flask. It
is dissolved in distilled water and made up to the mark. Effect of Type and Volume of Base
Effect of Concentrations of PNA and NaNO2 absorbance values are reproducible in the range 20–35°C.
However, colored solution was found to be unstable beyond
Absorbance was increased up to 0.2 M acid concentration in that temperature range. Hence, all the studies were carried
PNA solution and then decreased. Probably, more number out at room temperature.
of amine molecules occur in their ionic form at higher
concentration of acid, and hence, the rate of coupling is
Table 2: Effect of Time on coupling reaction
hampered. Higher intensity of color was found when volumes
of PNA (7.24 × 10−3 M) and NaNO2 (1.45×10−2 M) solutions Time (min) Absorbance*
were in the range of 0.8–1.0 mL. Persistent absorbance was 2 0.518
observed even at higher volumes of sodium nitrite, whereas 5 0.547
fluctuating results were observed with PNA [Figure 3]. 10 0.541
15 0.538
Effect of Time on Development of Color and Its 30 0.538
Stability
60 0.535
*At 8 µg/mL selexipag
Different time intervals (2–90 min) were chosen to study the
optimum time required for the formation (i.e., for coupling
reaction) and its stability of color at room temperature. Table 3: Effect of reactants addition sequence on
Absorbance values [Table 2] show that maximum intensity
absorbance
of color is achieved within 5 min and is stable almost up
to 1 h. Reactants and reagents Absorbance*
addition sequence
Drug+Base+DPNA 0.410
Sequence of Addition of Reagents
DPNA+Base+Drug 0.493
The effect of the sequence of addition of reagents on DPNA+Drug+Base 0.547
the formation of chromogen was studied. The observed *At 8 µg/mL selexipag. DPNA: Diazotized para nitroaniline
absorbance values [Table 3] indicate that the sequence
“diazotized pNA (DPNA) + Drug + Base” can be considered Table 4: Calibration values of selexipag
for the addition of reactants and reagents.[10]
Concentration (µg/mL) Absorbance*
Effect of temperature on colored complex stability was 2 0.131
inspected at various temperatures and found that the 4 0.259
6 0.408
8 0.547
10 0.682
12 0.816
*Average of three determinations
active hydrogen atom. The two possible substitution points Pyrazine is aromatic in character and has lower resonance
on selexipag are benzene ring and pyrazine ring. energy compared to benzene. It undergoes electrophilic
aromatic substitution on one of its resonance hybrids, which
are shown in Figure 5. From its canonical forms, four points
of electron deficient are visible at 2, 3, 5, and 6 positions[18]
indicating the possible attack by nucleophilic reagents at
those positions. As DPNA is considered as an electrophile,
the possibility of its attachment to pyrazine structure can
be eliminated. Hence, benzene ring is the only left over
option for attachment. To know more about the nature of
the formed dye (i.e., the number of DPNA substitutions on
the drug), reaction ratio between selexipag and DPNA was
determined.[19] Figure 6 shows that the ratio is 1:1, and hence,
Figure 6: Mole ratio method for complex formation at 8 µg/mL the scheme [Figure 7] illustrates the proposed mechanism.
of selexipag The substitution of DPNA is directed preferably to the para
position to the attached active group, and if para position is different amounts (50%, 100%, and 150%) of bulk samples
already occupied, then it will be directed to the ortho. Hence, of selexipag to 4 μg/mL to maintain the total amount of drug
DPNA is directed to the para position on the benzene ring (theoretical) concentration within the linearity range. The
which is further attached to pyrazine. percentage recovery values were in the range of 98.50–100.75
[Table 6]. A high level of accuracy for the proposed method
is evident as standard deviation (SD) values are lower and %
Validation of Proposed Method
relative SD (%RSD) values are <1.
The proposed was validated as per the existing ICH
Precision
guidelines.[20]
Intra- and inter-day precision were studied by selecting three
Linearity and range different concentrations of selexipag in the linear range
A graph was plotted between absorbance and concentrations (2–12 μg/mL). Of the six independent series, analysis was
(2–12 μg/mL) to obtain a linear calibration curve [Figure 8]. carried out on the same day and on 6 consecutive days for each
Three independent measurements were carried out for each concentration [Table 7]. Satisfactory precision of the method
concentration, and their mean value was indicated as a point of is evident from lower % RSD values in the range of 0.92–1.12
the calibration graph [Table 4]. The correlation coefficient of and 0.92–1.11, respectively, for intra- and inter-day studies.
linear regression equation (y = 0.069x−0.0095) was >0.999, and
hence, linearity of the proposed analytical method was tested. Ruggedness
Different optical and regression parameters are shown in Table 5. The developed method was evaluated for the ruggedness by
carrying out assay at different concentrations (2, 8, and 12 μg/
Accuracy mL) of selexipag by two different analysts on different days
Accuracy of the proposed method was tested by determining under the same optimized conditions. No significant difference
percent recovery values. This was performed by adding between the analyst values indicates the reproducibility of
results, and hence, the proposed method is rugged [Table 8].